Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency.

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

Biochemical thresholds for pathological presentation of ATP synthase deficiencies.

Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293 cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15-80% of controls by the knockdown of F1 subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10-30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.

TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.

Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP-synthase deficiency and leads to a neonatal mitochondrial encephalocardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70-/- cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1-deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate-limiting step of ATP-synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.-Kovalcíková, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nusková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliás, J., Cunátová, K., Korínek, V., Sedlacek, R., Mrácek, T., Houstek, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.

Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme.

The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150-400 kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.

Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice.

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6.

The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.

Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects.

Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.

The clinical, biochemical and genetic features associated with RMND1-related mitochondrial disease.

BACKGROUND: Mutations in the RMND1 (Required for Meiotic Nuclear Division protein 1) gene have recently been linked to infantile onset mitochondrial disease characterised by multiple mitochondrial respiratory chain defects. METHODS: We summarised the clinical, biochemical and molecular genetic investigation of an international cohort of affected individuals with RMND1 mutations. In addition, we reviewed all the previously published cases to determine the genotype-phenotype correlates and performed survival analysis to identify prognostic factors. RESULTS: We identified 14 new cases from 11 pedigrees that harbour recessive RMND1 mutations, including 6 novel variants: c.533C>A, p.(Thr178Lys); c.565C>T, p.(Gln189*); c.631G>A, p.(Val211Met); c.1303C>T, p.(Leu435Phe); c.830+1G>A and c.1317+1G>T. Together with all previously published cases (n=32), we show that congenital sensorineural deafness, hypotonia, developmental delay and lactic acidaemia are common clinical manifestations with disease onset under 2 years. Renal involvement is more prevalent than seizures (66% vs 44%). In addition, median survival time was longer in patients with renal involvement compared with those without renal disease (6 years vs 8 months, p=0.009). The neurological phenotype also appears milder in patients with renal involvement. CONCLUSIONS: The clinical phenotypes and prognosis associated with RMND1 mutations are more heterogeneous than that were initially described. Regular monitoring of kidney function is imperative in the clinical practice in light of nephropathy being present in over 60% of cases. Furthermore, renal replacement therapy should be considered particularly in those patients with mild neurological manifestation as shown in our study that four recipients of kidney transplant demonstrate good clinical outcome to date.

The mammalian homologue of yeast Afg1 ATPase (lactation elevated 1) mediates degradation of nuclear-encoded complex IV subunits.

Mitochondrial protein homeostasis is crucial for cellular function and integrity and is therefore maintained by several classes of proteins possessing chaperone and/or proteolytic activities. In the present study, we focused on characterization of LACE1 (lactation elevated 1) function in mitochondrial protein homeostasis. LACE1 is the human homologue of yeast mitochondrial Afg1 (ATPase family gene 1) ATPase, a member of the SEC18-NSF, PAS1, CDC48-VCP, TBP family. Yeast Afg1 was shown to mediate degradation of mitochondrially encoded complex IV subunits, and, on the basis of its similarity to CDC48 (p97/VCP), it was suggested to facilitate extraction of polytopic membrane proteins. We show that LACE1, which is a mitochondrial integral membrane protein, exists as part of three complexes of approximately 140, 400 and 500 kDa and is essential for maintenance of fused mitochondrial reticulum and lamellar cristae morphology. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4 (cytochrome c oxidase 4), COX5A and COX6A, and is required for normal activity of complexes III and IV of the respiratory chain. Using affinity purification of LACE1-FLAG expressed in a LACE1-knockdown background, we show that the protein interacts physically with COX4 and COX5A subunits of complex IV and with mitochondrial inner-membrane protease YME1L. Finally, we demonstrate by ectopic expression of both K142A Walker A and E214Q Walker B mutants, that an intact ATPase domain is essential for LACE1-mediated degradation of nuclear-encoded complex IV subunits. Thus the present study establishes LACE1 as a novel factor with a crucial role in mitochondrial protein homeostasis.

Autocrine effects of transgenic resistin reduce palmitate and glucose oxidation in brown adipose tissue.

Resistin has been originally identified as an adipokine that links obesity to insulin resistance in mice. In our previous studies in spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin (Retn) transgene specifically in adipose tissue (SHR-Retn), we have observed an increased lipolysis and serum free fatty acids, ectopic fat accumulation in muscles, and insulin resistance. Recently, brown adipose tissue (BAT) has been suggested to play an important role in the pathogenesis of metabolic disturbances. In the current study, we have analyzed autocrine effects of transgenic resistin on BAT glucose and lipid metabolism and mitochondrial function in the SHR-Retn vs. nontransgenic SHR controls. We observed that interscapular BAT isolated from SHR-Retn transgenic rats compared with SHR controls showed a lower relative weight (0.71 ± 0.05 vs. 0.91 ± 0.08 g/100 g body wt, P < 0.05), significantly reduced both basal and insulin stimulated incorporation of palmitate into BAT lipids (658 ± 50 vs. 856 ± 45 and 864 ± 47 vs. 1,086 ± 35 nmol/g/2 h, P ≤ 0.01, respectively), and significantly decreased palmitate oxidation (37.6 ± 4.5 vs. 57 ± 4.1 nmol/g/2 h, P = 0.007) and glucose oxidation (277 ± 34 vs. 458 ± 38 nmol/g/2 h, P = 0.001). In addition, in vivo microPET imaging revealed significantly reduced (18)F-FDG uptake in BAT induced by exposure to cold in SHR-Retn vs. control SHR (232 ± 19 vs. 334 ± 22 kBq/ml, P < 0.05). Gene expression profiles in BAT identified differentially expressed genes involved in skeletal muscle and connective tissue development, inflammation and MAPK and insulin signaling. These results provide evidence that autocrine effects of resistin attenuate differentiation and activity of BAT and thus may play a role in the pathogenesis of insulin resistance in the rat.

Sex difference in the sensitivity of cardiac mitochondrial permeability transition pore to calcium load.

Most of the experimental studies have revealed that female heart is more tolerant to ischemia/reperfusion (I/R) injury as compared with the male myocardium. It is widely accepted that mitochondrial dysfunction, and particularly mitochondrial permeability transition pore (MPTP) opening, plays a major role in determining the extent of cardiac I/R injury. The aim of the present study was, therefore, to analyze (i) whether calcium-induced swelling of cardiac mitochondria is sex-dependent and related to the degree of cardiac tolerance to I/R injury and (ii) whether changes in MPTP components-cyclophilin D (CypD) and ATP synthase-can be involved in this process. We have observed that in mitochondria isolated from rat male and female hearts the MPTP has different sensitivity to the calcium load. Female mitochondria are more resistant both in the extent and in the rate of the mitochondrial swelling at higher calcium concentration (200 µM). At low calcium concentration (50 µM) no differences were observed. Our data further suggest that sex-dependent specificity of the MPTP is not the result of different amounts of ATP synthase and CypD, or their respective ratio in mitochondria isolated from male and female hearts. Our results indicate that male and female rat hearts contain comparable content of MPTP and its regulatory protein CypD; parallel immunodetection revealed also the same contents of adenine nucleotide translocator or voltage-dependent anion channel. Increased resistance of female heart mitochondria thus cannot be explained by changes in putative components of MPTP, and rather reflects regulation of MPTP function.

Alteration of structure and function of ATP synthase and cytochrome c oxidase by lack of Fo-a and Cox3 subunits caused by mitochondrial DNA 9205delTA mutation.

Mutations in the MT-ATP6 gene are frequent causes of severe mitochondrial disorders. Typically, these are missense mutations, but another type is represented by the 9205delTA microdeletion, which removes the stop codon of the MT-ATP6 gene and affects the cleavage site in the MT-ATP8/MT-ATP6/MT-CO3 polycistronic transcript. This interferes with the processing of mRNAs for the Atp6 (Fo-a) subunit of ATP synthase and the Cox3 subunit of cytochrome c oxidase (COX). Two cases described so far presented with strikingly different clinical phenotypes-mild transient lactic acidosis or fatal encephalopathy. To gain more insight into the pathogenic mechanism, we prepared 9205delTA cybrids with mutation load ranging between 52 and 99% and investigated changes in the structure and function of ATP synthase and the COX. We found that 9205delTA mutation strongly reduces the levels of both Fo-a and Cox3 proteins. Lack of Fo-a alters the structure but not the content of ATP synthase, which assembles into a labile, ∼60 kDa smaller, complex retaining ATP hydrolytic activity but which is unable to synthesize ATP. In contrast, lack of Cox3 limits the biosynthesis of COX but does not alter the structure of the enzyme. Consequently, the diminished mitochondrial content of COX and non-functional ATP synthase prevent most mitochondrial ATP production. The biochemical effects caused by the 9205delTA microdeletion displayed a pronounced threshold effect above ∼90% mutation heteroplasmy. We observed a linear relationship between the decrease in subunit Fo-a or Cox3 content and the functional presentation of the defect. Therefore we conclude that the threshold effect originated from a gene-protein level.

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase.

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.

ROS production in brown adipose tissue mitochondria: the question of UCP1-dependence.

Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof- or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(-/-) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).

Mitochondrial ATP synthasome: Expression and structural interaction of its components.

Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities.

Effects of mtDNA in SHR-mtF344 versus SHR conplastic strains on reduced OXPHOS enzyme levels, insulin resistance, cardiac hypertrophy, and systolic dysfunction.

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mt(F344) conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mt(F344) conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractional shortening that was independent of blood pressure. In addition, conplastic rats exhibited reduced sensitivity of skeletal muscles to insulin action and impaired glucose tolerance. These results provide evidence that inherited alterations in mitochondrial genome, in the absence of variation in the nuclear genome and other confounding factors, predispose to insulin resistance, cardiac hypertrophy and systolic dysfunction.

The function and the role of the mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissues.

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is not included in the traditional textbook schemes of the respiratory chain, reflecting the fact that it is a non-standard, tissue-specific component of mammalian mitochondria. But despite its very simple structure, mGPDH is a very important enzyme of intermediary metabolism and as a component of glycerophosphate shuttle it functions at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism. In this review we summarize the present knowledge on the structure and regulation of mGPDH and discuss its metabolic functions, reactive oxygen species production and tissue and organ specific roles in mammalian mitochondria at physiological and pathological conditions.

Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations.

The loss of Surf1 protein leads to a severe COX deficiency manifested as a fatal neurodegenerative disorder, the Leigh syndrome (LS(COX)). Surf1 appears to be involved in the early step of COX assembly but its function remains unknown. The aim of the study was to find out how SURF1 gene mutations influence expression of OXPHOS and other pro-mitochondrial genes and to further characterize the altered COX assembly. Analysis of fibroblast cell lines from 9 patients with SURF1 mutations revealed a 70% decrease of the COX complex content to be associated with 32-54% upregulation of respiratory chain complexes I, III and V and accumulation of Cox5a subunit. Whole genome expression profiling showed a general decrease of transcriptional activity in LS(COX) cells and indicated that the adaptive changes in OXPHOS complexes are due to a posttranscriptional compensatory mechanism. Electrophoretic and WB analysis showed that in mitochondria of LS(COX) cells compared to controls, the assembled COX is present entirely in a supercomplex form, as I-III2-IV supercomplex but not as larger supercomplexes. The lack of COX also caused an accumulation of I-III2 supercomplex. The accumulated Cox5a was mainly present as a free subunit. We have found out that the major COX assembly subcomplexes accumulated due to SURF1 mutations range in size between approximately 85-140kDa. In addition to the originally proposed S2 intermediate they might also represent Cox1-containing complexes lacking other COX subunits. Unlike the assembled COX, subcomplexes are unable to associate with complexes I and III.

Compensatory upregulation of respiratory chain complexes III and IV in isolated deficiency of ATP synthase due to TMEM70 mutation.

Early onset mitochondrial encephalo-cardiomyopathy due to isolated deficiency of ATP synthase is frequently caused by mutations in TMEM70 gene encoding enzyme-specific ancillary factor. Diminished ATP synthase results in low ATP production, elevated mitochondrial membrane potential and increased ROS production. To test whether the patient cells may react to metabolic disbalance by changes in oxidative phosphorylation system, we performed a quantitative analysis of respiratory chain complexes and intramitochondrial proteases involved in their turnover. SDS- and BN-PAGE Western blot analysis of fibroblasts from 10 patients with TMEM70 317-2A>G homozygous mutation showed a significant 82-89% decrease of ATP synthase and 50-162% increase of respiratory chain complex IV and 22-53% increase of complex III. The content of Lon protease, paraplegin and prohibitins 1 and 2 was not significantly changed. Whole genome expression profiling revealed a generalized upregulation of transcriptional activity, but did not show any consistent changes in mRNA levels of structural subunits, specific assembly factors of respiratory chain complexes, or in regulatory genes of mitochondrial biogenesis which would parallel the protein data. The mtDNA content in patient cells was also not changed. The results indicate involvement of posttranscriptional events in the adaptive regulation of mitochondrial biogenesis that allows for the compensatory increase of respiratory chain complexes III and IV in response to deficiency of ATP synthase.

Impairment of adrenergically-regulated thermogenesis in brown fat of obesity-resistant mice is compensated by non-shivering thermogenesis in skeletal muscle.

OBJECTIVE: Non-shivering thermogenesis (NST) mediated by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) can be activated via the adrenergic system in response to cold or diet, contributing to both thermal and energy homeostasis. Other mechanisms, including metabolism of skeletal muscle, may also be involved in NST. However, relative contribution of these energy dissipating pathways and their adaptability remain a matter of long-standing controversy. METHODS: We used warm-acclimated (30 °C) mice to characterize the effect of an up to 7-day cold acclimation (6 °C; CA) on thermoregulatory thermogenesis, comparing inbred mice with a genetic background conferring resistance (A/J) or susceptibility (C57BL/6 J) to obesity. RESULTS: Both warm-acclimated C57BL/6 J and A/J mice exhibited similar cold endurance, assessed as a capability to maintain core body temperature during acute exposure to cold, which improved in response to CA, resulting in comparable cold endurance and similar induction of UCP1 protein in BAT of mice of both genotypes. Despite this, adrenergic NST in BAT was induced only in C57BL/6 J, not in A/J mice subjected to CA. Cold tolerance phenotype of A/J mice subjected to CA was not based on increased shivering, improved insulation, or changes in physical activity. On the contrary, lipidomic, proteomic and gene expression analyses along with palmitoyl carnitine oxidation and cytochrome c oxidase activity revealed induction of lipid oxidation exclusively in skeletal muscle of A/J mice subjected to CA. These changes appear to be related to skeletal muscle NST, mediated by sarcolipin-induced uncoupling of sarco(endo)plasmic reticulum calcium ATPase pump activity and accentuated by changes in mitochondrial respiratory chain supercomplexes assembly. CONCLUSIONS: Our results suggest that NST in skeletal muscle could be adaptively augmented in the face of insufficient adrenergic NST in BAT, depending on the genetic background of the mice. It may provide both protection from cold and resistance to obesity, more effectively than BAT.